In order to optimize the genetic transformation system for Malus baccata Borkh., DNA recombination technology was used for inserting green fluorescence protein gene (GFP) to pBI121, and then a new recombinant plasmid pBI121-eGFP was constructed. The plasmid was transferred to M. baccata Borkh. after introduced into Agrobacterium tumefaciens EHA105. Results showed that the efficiency was higher in the conditions described as follows: Excised leaves were inoculated with A. tumefaciens for 5~8 min, washed by MS containing 500 mg/L Cef and transferred to co-culture medium for three days, and then placed on selection medium containing 300 mg/L Cef and kan increased from 5 mg/L to 20 mg/L. Seven resistant plants were acquired in transgenic process and detected by PCR, RT-PCR, green fluorescence observation and protein signal analysis. Six of the seven transgenic plants were verified to be positive in PCR detection, green fluorescence and fluorescence protein signal in three of the six plants leaves (T1, T3 and T6) was stronger. The results of the molecular detection and fluorescence detection were the same, so it is possible to find transgenic plant by detecting the green fluorescence directly and achieves the goal for optimizing genetic transformation system for Malus baccata Borkh.

Newcastle diseases (ND) is one of the two serious diseases of poultry, Newcastle diseases virus (NDV) is the pathogen of the disease. To understand the molecular properties, pathogenicity of wildfowl NDV and its relationship with poultry NDV, a NDV strain, NDV2, was isolated from wildfowl in 2011 and confirmed virulent using standard biological virulence tests. Nine pairs of specific primers were designed from sequence of NDV deposited in GenBank, and the complete genomic sequence of NDV2 strain was 15 192 nt(GenBank accession No. KF306265) and its genome comprised six ORFs, namely, encoded nucleocapsid protein(NP), phosphoprotein(P), matrix protein(M), fusion protein(F), haemagglutinin-neuraminidase(HN) and large protein(L). The lengths of each gene were respectively 1 753, 1 451, 1 241, 1 792, 2 004 and 6 703 nt. NDV2 was classifid as ClassⅡ, genotype Ⅶ. Comparison of the homologies of each protein showed that NDV2 was highly homologous to several domestic genotype Ⅶ strains, such as Egret.GX.11, Duck.WF.00 and Goose.GD450.11 in recent years, which was far higher than strains of other regions and other genotypes.These results suggested that NDV2 had high affinity to domestic strains. The JS10 strain of ClassⅠ was the least homologous to NDV2. Sequence analysis of the F gene showed that 15 192 nt strains (including NDV2 strain) had a common motif for RRQKRF or KRQKRF which was consistent with highly pathogenic NDVs. F and HN genotyping showed consistency between NDV2 and the majority of the reference strains. However, the Indian. NDV4 strain from chicken, it belonged to genotypeⅡfor F gene, but for HN gene it belonged to genotype Ⅶ. Because NDV2 had start sequences identical to those of other reference strains, its start sequences were also highly conserved. The NP, P, M and L termination sequences of NDV2 were consistent with those of the reference strains but differed from those of the F and HN genes. NDV2 had termination sequences identical to those of La Sota and Sweden95 but different from those of other field strains. To better understand the pathogenicity of NDV2 in different hosts, we used NDV2 to attack specific pathogenfree(SPF) chicken(Gallus gallus), duck(Anas platyrhynchos platyrhynchos Linnaeus.) and pigeon(Rupestris Pallas); each attack group included a cohabitation infection group. After attack, the three hosts developed clinical symptoms at different times. Dissection of the infected hosts showed intestinal or tracheal bleeding and glandular stomach congestion. The pathogenicity of NDV2 in SPF chicken and pigeon was higher than that in duck; the strain further caused 50% and 20% cohabitation rates in infected chicken and pigeon. This research provides theoretical data for genetic variation characteristics and pathogenic rule of NDV.

Rice starch viscosity (RVA profile) characteristics can reflect the differences of starch quality of different varieties of rice sensitively, its genetic performance and the influence of starch synthesis-related genes(SSRGs) on RVA profile had received extensive attention. In this paper, we used backcross inbred lines as materials which were constructed by indica PTGMS line Guangzhan 63S and rice potential restorer line CG132R and composed of different allele combinations of soluble starch synthase Ⅱa gene (SSII-3) and granule-bound starch synthase gene (Wx) to study the influence of rice SSII-3 gene on RVA profile characteristics in non-glutinous rice, and compare the influence of SSII-3 gene with Wx gene on RVA profile characteristics. Main results were as follows:(1)gelatinization temperature had significant or extremely significant correlations with seven RVA profile characteristics except peak viscosity(PKV). It had significant or extremely significant negative correlations with hot paste viscosity(HPV), cool paste viscosity(CPV), consistence(CSV), setback(SBV) and peak time(PeT), but had extremely significant positive correlations with breakdown(BDV) and pasting temperature(PT). It indicated that SSII-3 gene had important influence on RVA profile characteristics in non-glutinous rice, and the influence being in the opposite direction compared with Wx gene. (2)Effect of SSII-3 gene on RVA profile characteristics was influenced because of the difference in Wx genotype. Split block design result showed that interaction between SSII-3 and Wx gene had significant or extremely significant influence on hot paste viscosity(HPV), cool paste viscosity(CPV) and consistence(CSV). When the Wx genotype of rice was TT(WxbWxb) or GT(WxaWxb), the consistence (CSV) of rice starch presented decreasing trend with the increase of gelatinization temperature, however, when Wx genotype of rice was GG(WxaWxa), consistence (CSV) had no regular variation trend. (3)Pasting temperature(PT) and peak time(PeT) of RVA profile characteristics were mainly affected by SSII-3 gene, however, peak viscosity(PKV), cool paste viscosity(CPV), hot paste viscosity(HPV), consistence(CSV), setback(SBV) and breakdown(BDV) were mainly affected by Wx gene. As the major gene controlling the gelatinization temperature of rice, SSII-3 gene is another important gene for RVA profile besides Wx gene, studying the influence of SSII-3 gene on RVA profile in non-glutinous rice and the interaction with Wx gene has important application value in evaluation of rice cooking and eating quality and breeding of high quality rice varieties.

1-deoxy-D-xylulose 5-phosphate reductoisomerase (DXR) is the key enzyme of terpenoid metabolic pathway in 2-C-Methyl-D-erythritol 4-phosphate (MEP) pathway in plant, and its expression level affects the contents of terpenoid. A new putative gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase (designated as TwDXR), which catalyses the second step in the 1-Deoxy-D-xylulose 5-phosphate (DXP) biosynthetic pathway, which converts DXP to 2-C-Methyl-D-erythritol 4-phosphate (MEP), as an important role in regulating the MEP pathway was isolated from young leaves of Tripterygiun wilfordii by RT-PCR (reverse transcription polymerase chain reaction) and rapid amplification of cDNA ends (RACE) for the first time. The full-length cDNA of the putative TwDXR was 1 674 bp containing a 1 410 bp open reading frame (ORF) encoding a polypeptide of 469 amino acids (GenBank accession No. KJ174341). Comparative and bioinformatic analysis showed that TwDXR had extensive homology with DXRs from other plant species, such as Populus trichocarpa. Tissue expression pattern analysis revealed that the putative TwDXR and 3-hydroxy-3-methylglutaryl-CoA reductase gene (TwHMGR) which catalyzed the conversion HMG-CoA to mevalonate (MVA), were constitutively expressed in all the tested tissues. The expression of TwDXR was strong in leaf, and TwHMGR was strong in adventitious root. The putative TwDXR and TwHMGR was found to be an elicitor-responsive gene, which could be induced by AgNO3(Ag+) and methyl jasmonate (MeJA). TwDXR was highest on 4th day, and TwHMGR was highest on 2nd day after inducing by 30 mmol/L AgNO3(Ag+). TwDXR and TwHMGR were highest on 2nd day after inducing by 50 mmol/L methyl jasmonate (MeJA).The highest accumulation of the triptolide was on 4th day and and wilforine was on 6th day after 30 mmol/L AgNO3(Ag+), respectively. The triptolide and wilforine were induced and accumulated highest content on 6th day after 50 mmol/L MeJA. These results will help for understanding the role of DXR and HMGR in isoprenoid biosynthesis, especially in promoting the content of active secondary metabolites in T. wilfordii.

Flavanone 3-hydroxylase (F3H) is a key enzyme in the metabolic pathways of flavonoid compounds in plants. The open reading frame(ORF) of F3H gene, which included 1 107 basic group and encoded a 368 amino acids protein, was cloned from tea (Camellia sinensis) by RT-PCR. The deduced protein molecular weight was 41.46 kD and its theoretical isoelectric point was 5.61. The results of Real-time fluorescent quantitative PCR showed that CsF3H had high expression levels in the leaves, and was influenced by light condition. The gene was cloned into the expression vector PRSF for expression in prokaryotic cells. The SDS-PAGE results showed that the F3H peotein was expressed in Escherichia coli BL21. The optimal inducing conditions including temperature, isopropyl-β-d-thiogalactoside(IPTG) concentration and time were studied , and were 28 ℃, 1.0 mmol/L, 5 h, respectively. The activity of the recombinant protein in vitro was tested by high performance liquid chromatography(HPLC) method. Experiment results showed that purified protein had the F3H enzyme activity and transfered naringenin(N) and eriodictyol(E) respectively to dihydrokaempfero(DHK) and dihydroquercetin(DHQ), while the recombinant protein had a much more preference for eriodictyol(E). The research provides basic data for the kinetic study and organ specificity of CsF3H protein.

Maternally inherited mitochondrial DNA (mtDNA) has been used extensively to determine genetic diversity, guide genetic resource conservation, and determine phylogeny. Zaosheng cattle, mainly produced in Qingyang Prefecture of Gansu, were introduced to Pingliang, Guyuan, and other areas coinciding with the growth of farming culture. Local genetic groups in various locations were formed through long term domestication and adaptation, respectively. To investigate the genetic diversity of Zaosheng cattle, six Zaosheng cattle groups (Qingyang group, QZ; QZ×South Devon group, QZ×N; Pingliang group, PZ; PZ×Simmental, PZ×X; Guyuan group, GZ; GZ×Qinchuan cattle, GZ×Q) were analyzed using mtDNA D-loop. A total of 131 variable sites and 103 haplotypes were identified. Among the QZ, PZ and GZ groups, the average number of nucleotide differences (13.884, 10.266, 3.111, respectively) and nucleotide diversity (0.022 95, 0.016 82, 0.005 11, respectively) decreased. Similar results were observed in QZ×N , PZ×X and GZ×Q groups (15.362, 9.264, 7.495, respectively; and 0.025 31, 0.015 24, 0.012 33, respectively for nucleotide differences and diversity). There were 6 shared and 32 unique haplotypes found in the QZ group, which was greater than PZ(6 shared, 9 unique)and GZ (2 shared, 5 unique). In conclusion, the genetic diversity of QZ and QZ×N was higher than other groups, and the genetic diversity of crossbred groups was similar to native groups. Consequently, crossbreeding had little influence on mtDNA genetic diversity in Zaosheng cattle and crossbreeding of Zaosheng cattle was generally through paternal crosses. We can also draw a conclusion that Zaosheng cattle in Pingliang and Guyuan could have migrated from Qingyang and gradually formed two maternal branches and provide a scientific foundation for genetic resources conservation of Zaosheng native cattle.

Hair follicle is a appendages of skin, and its periodic regeneration involves the activation and regulation of hair follicle stem cells. Cashmere goats (Capra hircus) skin tissue samples of different month age, was fixed, dehydrated, embeded, paraffined and sliced, which was finally stained by hematoxylin and eosin stain(HE). Three antibodies of keratin 15(K15), tyrosine-related protein 2(Trp2) and antibody of proliferating cell nuclear antigen 3(PCNA3) were used to determine the niche of hair follicle stem cells by immunohistochemistry. The results showed that Trp2 expressed in bulge of hair follicle in 2 kinds of hair follicle, but the signal was stronger in primery hair follicles; K15 mainly expressed outer root sheath under of bulge in telogen and expressed from under bulge to half of both hair folllicles; PCNA strongly expressed in anagen hair follicle but expressed weaker in telogen. Trp2 provides the site of the stem cell niche include diffirent stem cell types in hair follicles, K15 illustrates the migration trajectory of stem cell, PCNA reflects the propagation of hair follicle stem cells. All of the findings supply the foundation of finding the derect marker of hair follicles and regulation mechanism of hair follicle stem cells during the cashmere wool regeneration in Cashmere goats.

The tumor necrosis factor (TNF) and tumor necrosis factor receptor (TNFR) superfamilies play crucial roles in both innate and adaptive immunity. In this study, the fragment of TNFR (LcTNFR) was identified from the EST(expressed sequence tag)database of large yellow croaker (Larimichthys crocea), and the full length cDNA of LcTNFR of 2 016 bp was obtained using SMART-RACE method, consisting of a 5' UTR of 64 bp, an ORF of 1 350 bp and a 3' UTR of 602 bp(GenBank accession No KF432416). The deduced protein was composed of 449 amino acids, with a predicted pI of 5.47 and an estimated molecular mass of 49.7 kD. Protein structure prediction by SWISS-MODEL found that LcTNFR contained 6 α-helix and 0 β-fold. Blast analysis results revealed that LcTNFR had the highest identity with Maylandia zebra 10B-like (74%), followed with Danio rerio ovarian TNFR (51%) and with Carassius auratus TNFR Ⅰ(47%). Phylogenetic tree of the TNFR amino acid sequences analysis furtherly indicated that LcTNFR together with TNFR 10 B - like, TNFR typeⅠ and ovarian TNFR formed a branch. The results of quantitative Real-time PCR (qRT-PCR) showed that LcTNFR was expressed ubiquitously in examined tissues of male and female fishes. The expression level of LcTNFR was significantly higher in ovary than that in other tissues including testis, and the expression level of LcTNFR in spleen, which is an organ involved in immune function, was also higher than that in other examined tissues. In the spleen, the expression level of LcTNFR was up-regulated significantly (P < 0.05) at the 2 day and 4 day after Vibrio parahaemolyticus challenge, and then it restored to the control level at 8 day. The results may provide insight into the immunology research and the development of disease-resistance genetic markers for large yellow croaker.

Na+/K+-ATPase α1 is a key enzyme in regulating the osmotic pressure of aquatic animals. In this study, the cDNA of Na+/K+-ATPase α1 gene was first cloned from Chinese mitten crab (Eriocheir sinensis) using RT-PCR and RACE. The full length of cDNA was 3 805 bp (GenBank No. KC691291.1). It contained a 306 bp 5' UTR and 388 bp 3' UTR, as well as a 3111 bp open reading frame encoding 1 037 amino acids. The amino acid sequence shared as high as 91.9%~ 98.5% homology with other aquatic crustaceans, indicating its high conservation in crustaceans. Quantitative RT-PCR indicated the highest expression level in the hepatopancreas, followed by the gill, the intestines and stomach, and then the muscle and heart. Na+/K+-ATPase α1 expression was detected at the eight developmental stages of Eriocheir sinensis; but a low, insignificant expression was found in the five zoea stages (zoea Ⅰ to zoea Ⅴ). Surprisingly, expression reached the highest level at the megalopa stage and then significantly decreased at the larva and juvenile stages (P<0.05). Culturing the adult crabs in brackish water (salinity 21) and fresh water for 30 days yielded the following results: Expression in the muscle and hepatopancreas of crabs reared in brackish water was significantly higher (P<0.05) than that of crabs reared in fresh water; expression in the gill in fresh water was significantly higher (P<0.05) than that in brackish water; and finally, expression in the gonad did not significantly differ between brackish and fresh waters (P>0.05). These results showed that the hepatopancreas and gill were the dominant organs of Na+/K+-ATPase α1 gene expression in Eriocheir sinensis, indicating that this gene played an important role in regulating the osmotic pressure of the species. This study provides based data for research on suitable salinity control in reproduction, zoea production, and parental conservation of crab.

Thioredoxin interacting protein (Txnip) is a multifunctional protein involved in many cellular and physiological processes, and can regulate cellular redox state by inhibiting the activity of thioredoxin. To explore effects of glucose and adenosine-containing molecule on expression of Txnip mRNA and regulation pathway for transcription in porcine adipocytes, the preadipocytes isolated from subcutaneous adipose tissue obtained from 3~7 days old piglets(Sus scrofa) were cultured and induced to differentiate into mature adipocytes, in which carbohydrate response element binding protein gene (ChREBP) was silenced by transfection of ChREBP-RNAi expression plasmid. Non-transfected and transfected adipocytes were cultured in medium with 0, 5 or 15 mmol/L glucose with or without NAD+ for 24 h. Expression level of Txnip and ChREBP mRNA was determined by fluorescence Real-time PCR. The results showed that ChREBP mRNA expressions were up-regulated 4.57 and 6.69 times (P＜0.01) in porcine adipocytes cultured in medium with 5 mmol/L or 15 mmol/L glucose, respectively, but there was no difference (P＞0.05) in the cells cultured in same glucose concentrations of medium with or without NAD+, which indicated NAD+ alone or accompanied by glucose have no effect on the ChREBP mRNA expression. Expression of Txnip mRNA was increased by glucose stimulation (P＜0.01). Txnip mRNA expression was not elevated by NAD+ under glucose-free condition (P＞0.05), but increased significantly in the presence of glucose (P＜0.01). In ChREBP-silencing adipocytes, levels of Txnip mRNA expression had no difference (P＞0.05) and slightly increased by glucose (P＜0.05; ~1.34 times) compared with non-transfected adiocytes cultured in glucose-free medium. However, this stimulation of glucose on Txnip mRNA expression was reduced 82% and 91% in ChREBP-silencing adipocytes cultured in medium with 5 mmol/L and 15 mmol/L glucose than that in non-transfected cells cultured under the same condition, respectively. NAD+ did not further induce Txnip mRNA expression on the basis of glucose stimulation (P＞0.05), and stimulation of NAD+ on Txnip mRNA expression reduced 91% and 93% (P＜0.01) in ChREBP-silencing adipocyes than that in non-transfected cells cultured in medium with 5 mmol/L and 15 mmol/L glucose, respectively. These results suggested that NAD+ induces Txnip transcription expression dependent on glucose, and the stimulation of glucose and adenosine-containing molecule on Txnip mRNA expression were mediated by ChREBP. This study can provide useful data for research on the role of ChREBP in glucose and lipid homeostasis.

Tomato (Solanum lycopersicum L.) is a very important commercial crop and also a significant model for studying the floral development in a sympodial perennial plant. Broadly speaking, flower development can be divided into four steps: flowering transition, meristem identity decision, floral organ initiation and floral organ morphogenesis. In recent years, our understanding with the process of floral development in tomato has improved considerably, but there are few reviews about this. By reviewing researches about floral development in tomato in recent years, the morphology character and gene regulation during the process of floral development in tomato were summarized. This review focused on the genes involved in the flowering transition and meristem identity, as well as the interrelation of them, in order to provide some theoretical basises for the further research about these two processes in tomato.

Genetically modified organisms detection reference materials(GMOs detection RMs) used in genetically modified organisms (GMOs) detection are essential for GMOs safety supervision, qualitative and quantitative detection. To date, only a few institutions, such as EU Joint Research Centre (Institute for Reference Materials and Measurements, IRMM) and American Oil Chemists' Society (AOCS), have developed certified reference material (CRM) for GMOs testing. In China, research and development of GMOs detection RM are still at the initial stage. Based on the price of CRMs on GMOs testing is very expensive, and the available of CRMs for GMOs can not meet requirements in China, the funding agency in China supported a research project named "Study on RMs for GMOs detection", which aims to develop CRMs for GMOs detection used in Chinese GMOs safety supervision requirements. This paper presented overview on production process and value characterization pattern of current existing RMs based on matrix, genomic DNA and plasmid DNAs. We also addressed the problems in development of RMs of GMOs detection in this paper. This review can provide a useful reference for production of Chinese RMs of GMOs detection, and theoretical basis for value characterization and uncertainty evaluation of RMs.

To establish the detection method of genetically modified (GM) cotton event GHB119, which has not been authorized by the Ministry of Agriculture of China, the specific primer pair and probe were designed based on the 3'flanking sequence between the cotton genome and inserted exogenous fragment of GHB119 for protecting and improving the implementation of laws and regulations for GM products safety. After optimizing the reaction conditions, a novel event-specific quantitative Real-time PCR method for GHB119 detection was established. The applicability of the method was determined. Results showed that the developed Real-time PCR method was specific for GM cotton GHB119 detection, the limit of detection (LOD) and quantification (LOQ) were 10 and 25 copies of GHB119 haploid genomic DNA, respectively. Results of the quantification test of GM ingredient in blind samples showed that the bias between the true value and the measured value, the standard deviation (SD) and the relative standard deviation (RSD) were all in the acceptable range. Therefore, the established event-specific quantitative Real-time PCR method for GM cotton GHB119 detection has high specificity and good sensitivity, and is suitable for quantification of GHB119 ingredient in mixed samples quickly and accurately.

In order to study multi-gene interaction and reduce the blindness of many transformation, CaMV 35S promoter and nos terminator were added into gene del and ros upstream and downstream, respectively. del and ros genes can regulate anthocyanin synthesis. Intermediate vector pBI121-del and pCAMBIA1301-ros were constructed. BamHⅠand BglⅡproduced the same end tetra-nucleotide segments. The CaMV 35S promoter separately driven del and ros genes were constructed in plant expression vector pCAMBIA2301 under ligase. Strawberries(Fragaria ananassa Duch.) were treated by the constructed vector pCAMBIA2301-del-ros via Agrobacterium tumefaciens LBA4404 mediated transformation method. Transgenic strawberry lines were screened by PCR analysis. Observed by organizations, the roots and leaves of transgenic strawberry lines became reddish purple. The expression of anthocyanin biosynthetic genes anthocyanidinsynthase(ANS), chalcone-flavanone isomerase(CHI), flavanone 3-hydroxylase(F3H), dihydroflavonol-4-reductase(DFR) and UDP glucose-flavonoid 3-O-glcosyl-transferase(UFGT) were all enhanced in transgenic strawberry plant. The results were further confirmed that the plant expression vector pCAMBIA2301-del-ros could be used to transform plants. Construction of plant expression vector pCAMBIA2301-del-ros can provides a good idea for constructing a multi-gene plant expression vector and a method for studying interaction of multiple genes.

In order to develop and verify a high sensitivity sandwich enzyme-linked immunosorbent assay (ELISA) for detection of neomycin phosphotransferase (NPTⅡ) as a novel method for the quantitative determination of genetically modified (GM) cotton(Gossypium sp.), NPTII protein was prepared as immunogen in vitro, the sandwich ELISA based on the rabbit(Lepus) polyclonal serum (as capture antibody) and Mus musculus monoclonal antibody (mAb) (as detecting antibody) was developed. The standard curve was established with series content NPTⅡ protein as the standard. Each index of the method was validated and the quantitative determination of NPTII in GM cotton was carried out by this method. The linear range of the sandwich ELISA was 3.370~108.125 ng∕mL with a detection limit of 1.97 ng/ml for NPTⅡ. The recoveries of NPTⅡ from non-GM cotton proteins ranged 96.11%~104.69%, respectively, with a coefficient of variation (CV) of 6.58%~7.33%. The sandwich ELISA method had no cross reaction with non-GM cotton endogenous proteins and other types of transgenic protein. After applying this method, we found that the NPTⅡ expression levels of various parts in transgenic cotton plant seedling were significantly different. The results showed that this sandwich ELISA can be used as a specific and reliable immunoassay for detecting NPTⅡ content in GM cotton, which can provide references for establishing the GM crops detection methods and biological safety evaluation, with good application value and prospects.